Disorders of lipid metabolism are associated with various types of diseases including diabetes and cardiovascular diseases (CVD). Blood concentrations of lipids, such as triglycerides and cholesterol, are known to provide indications of the abnormal lipid metabolism. However, the concentration of cholesterol carried by lipoproteins, such as high-density lipoprotein (HDL), does not always reflect the presence and risk of these diseases. Not only quantitative indices (such as the concentration of cholesterol) but also qualitative indices focused on the functionality of lipoproteins have attracted attention.
For example, several reports have shown that some HDL cholesterol (HDL-C) raising agents, such as CETP inhibitors, did not reduce the risk of CVD even though they effectively increased the blood concentration of HDL-C. This indicates the possibility that the concentration of HDL-C does not completely reflect the risk of CVD. Recently, attention has focused on the physiological functions of high-density lipoprotein (HDL) to monitor the risk of CVD. It has been reported that the HDL's capacity to excrete cholesterol from the peripheral tissue is a negative prognostic factor for the risk of CVD.
As the method of examining the functionality of HDL, a method of using fluorescence-labeled cholesterol and cultured cells is described in, for example, Sankaranarayanan S. et. al, A sensitive assay for ABCA1-mediated cholesterol efflux using BODIPY-cholesterol. J. Lipid Res., vol. 52, p. 2332-2340 (2001). In this method, functional level of HDL to promote cholesterol excretion from cells is examined by measuring the removal amount of fluorescence-labeled cholesterol from the macrophages containing the labeled cholesterol by HDL. This method includes the following four steps: (1) incorporating labeled cholesterol into macrophage cells; (2) adding an inhibitor for acyl-CoA: cholesterol acyltransferase to inhibit the esterification of cholesterol in the cells; (3) adding HDL to stimulate the macrophages; and (4) recovering the culture supernatant and cell lysate and quantifying the labeled cholesterol in these liquids.
On the other hand, PCT International Publication No. WO 2012/104411 describes a method of determining the presence of dyslipidemia by fluorescence-labeled cholesterol without using cultured cells. In this method, the peripheral monolayers of various lipoproteins including low-density lipoprotein (LDL), HDL or the like is labeled with fluorescence-labeled cholesterol (cholesterol pyrene), and it is determined whether a subject has dyslipidemia based on the fluorescence spectra obtained by measuring the labeled lipoproteins. The literature describes that the labeled lipoproteins can be separated from fluorescence-labeled free cholesterol by ultracentrifugation, dialysis or FPLC using a gel-filtration column.